fluorotryptophan as a novel PET probe for the visualization of

Fig.1 Tryptophan (Trp) and metabolites of tryptophan (1–10). Tryptophan metabolism is ...... The filter cake was carefully washed with an excess of...
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Discovery of 7‑[18F]Fluorotryptophan as a Novel Positron Emission Tomography (PET) Probe for the Visualization of Tryptophan Metabolism in Vivo Boris D. Zlatopolskiy,*,†,‡,§ Johannes Zischler,†,‡,# Dominique Schaf̈ er,†,# Elizaveta A. Urusova,†,‡,§ Mehrab Guliyev,†,‡ Olesia Bannykh,†,‡ Heike Endepols,†,‡,§,⊥ and Bernd Neumaier*,†,‡,§ †

Institute of Neuroscience and Medicine, INM-5: Nuclear Chemistry, Forschungszentrum Jülich GmbH, Jülich 52428, Germany Institute of Radiochemistry and Experimental Molecular Imaging, University Clinic Cologne, Cologne 50937, Germany § Max Planck Institute for Metabolism Research, Cologne 50931, Germany ⊥ Department of Nuclear Medicine, University Clinic Cologne, Cologne 50937, Germany ‡

S Supporting Information *

ABSTRACT: Tryptophan and its metabolites are involved in different physiological and pathophysiological processes. Consequently, positron emission tomography (PET) tracers addressing tryptophan metabolic pathways should allow the detection of different pathologies like neurological disorders and cancer. Herein we report an efficient method for the preparation of fluorotryptophans labeled in different positions with 18F and their biological evaluation. 4−7-[18F]Fluorotryptophans ([18F]FTrps) were prepared according to a modified protocol of alcohol-enhanced Cu-mediated radiofluorination in 30−53% radiochemical yields. In vitro experiments demonstrated high cellular uptake of 4−7-[18F]FTrps in different tumor cell lines. 4, 5-, and 6-[18F]FTrps, although stable in vitro, suffered from rapid in vivo defluorination. In contrast, 7-[18F]FTrp demonstrated a high in vivo stability and enabled a clear delineation of serotonergic areas and melatonin-producing pineal gland in rat brains. Moreover 7-[18F]FTrp accumulated in different tumor xenografts in a chick embryo CAM model. Thus, 7-[18F]FTrp represents a highly promising PET probe for imaging of Trp metabolism.

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INTRODUCTION Tryptophan (Trp) (Figure 1) is an essential proteinogenic amino acid that contains an indole ring in the side chain. Trp cannot be synthesized by mammals and must be obtained from external sources.1 Tryptophan is the least abundant amino acid in animal proteins (∼1.3%) and serves mainly as a precursor for various metabolic pathways that result in different important biomolecules such as serotonin (1), melatonin (2), niacin (vitamin B3, 3), and kynurenines (4−8) (Figure 1).2−4 The kynurenine pathway accounts for the catabolism of at least 95% of ingested tryptophan and is closely related to the immune system. Its functions range from immunotolerance and neuroprotection to inflammation and oxidative stress.5 The final product of the kynurenine pathway is nicotinamide adenine dinucleotide (NAD+, 9), an important coenzyme utilized by all living cells. However, in the healthy brain, the neuroactive metabolites (e.g., kynurenic acid and quinolinic acid) are present only in the low nanomolar range, 100−1000 times lower than the tryptophan concentration, and are probably inactive.6,7 At the same time the © 2017 American Chemical Society

concentration of serotonin and its metabolite 5-hydroxyindoleacetic acid (10) in the brain amounts to approximately one-fifth of that of tryptophan.6 Serotonin (1) is an important neurotransmitter involved in many processes throughout the brain. Tryptophan metabolism is altered in numerous pathological processes in the brain, such as neuropsychiatric and neurodegenerative diseases as well as in peripheral organs and tissues, e.g., in cancer and diabetes. These impairments make tryptophan metabolism an attractive target for molecular imaging of different pathologies. PET imaging of Trp metabolism using 11C-labeled Trp analogues such as α-[11C]methyl-L-tryptophan ([11C]AMT, [11C]11)8,9 and 5-hydroxy-L-β-[11C]tryptophan ([11C]HTP, [11C]12) (Figure 2)10,11 has been shown to improve the accuracy of primary diagnostics, staging and therapy and followup of tumors and neurological disorders.6,12−20 Nevertheless, the Received: August 24, 2017 Published: October 20, 2017 189

DOI: 10.1021/acs.jmedchem.7b01245 J. Med. Chem. 2018, 61, 189−206

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Figure 1. Tryptophan (Trp) and metabolites of tryptophan (1−10).

Figure 2. Clinically applied 11C-labeled Trp analogs ([11C]11 and [11C]12), [18F]FETrp ([18F]13) and [18F]fluorotryptophans ([18F]14−17, [18F]FTrps) prepared and tested in this study.

short half-life of 11C (t1/2 = 20 min), as well as the laborious and time-consuming procedures for the preparation of these tracers has hampered the implementation of Trp-PET in clinical practice. In contrast, the longer half-life of 18F (t1/2 = 109 min) should enable the development of 18F-labeled tryptophans for the visualization of Trp metabolism. The first procedure for the preparation of racemic 5- and 6-[18F]fluorotryptophans via pyrolysis of tetrafluoroborates of the corresponding diazonium salts in the presence of [18F]HF was published as early as 1972 (Table 1).21 However, low molar activities (0.5 MBq/μmol), low RCYs, long preparation times, and the potentially explosive nature of diazonium salts prevented the further development of this method. Generally, [18F]Trps are often very difficult to synthesize using conventional radiofluorination procedures. Recently, (S)-N-(2-[18F]fluoroethyl)tryptophan ([ 18 F]FETrp, [18F]13; Figure 2) was tested as indoleamine 2,3dioxygenase 1 selective PET probe. Indoleamine 2,3-dioxygenases (IDO1 and -2) and tryptophan 2,3-dioxygenases (TDO1 and -2) catalyze the first and rate-limiting step of the oxidative opening of the indole ring in the tryptophan catabolism via the

Table 1. Optimization of Alcohol Enhanced Cu-Mediated Radiofluorination with Respect to 18F− Recovery and Precursor Amount Using 33 as a Model Substratea 32 (μmol) Et4NHCO3 (μmol) Cu(py)4(OTf)2 (μmol) nBuOH/DMA (μL/μL) drying time (min) loss on QMA (%) loss on reaction vial (%) RCC (%)

20 4.33 10 500/250 2−5 2.0 ± 0.5 5±3 91.9 ± 2.7

10 2.16 5 500/250 2−6 3.0 ± 0.5 7±2 89.7 ± 4.5

5 1.1 2.5 300/150 3−5 7.0 ± 3 13 ± 3 82.4 ± 7.6

a Conditions: elution of 18F− (100−200 MBq) with Et4NHCO3 in MeOH (500 μL), MeOH evaporation, addition of a solution of 33 and Cu(py)4(OTf)2 in nBuOH/DMA, 110 °C, 15 min, RCCs were determined by radio-TLC after addition of H2O (1 mL). All experiments were carried out in triplicate.

kynurenine pathway.22−24 Henrottin et al.25 demonstrated recently the selective and prolonged retention of [18F]13 in 190

DOI: 10.1021/acs.jmedchem.7b01245 J. Med. Chem. 2018, 61, 189−206

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Figure 3. Synthesis of precursor 18 for the preparation of 4-[18F]FTrp ([18F]14).

Figure 4. Synthesis of precursors 26 and 28 for the preparation of 5-[18F]FTrp ([18F]15). Additionally substrate 2936 for the synthesis of 6-[18F]FTrp ([18F]16) is shown.

MBq 18F−) using a cumbersome three step preparation procedure. It consisted of 18F/19F isotopic exchange followed by Rh-catalyzed reductive decarbonylation of the intermediate radiolabeled aldehyde and deprotection affording 4-[18F]FTrp in 13% RCY within 115 min. The desired tracer was obtained with excellent radiochemical and enantiomeric purity (>99% ee) but with low molar activity of >0.07 MBq/μmol (Table 1). Fortunately, the discovery of Cu-mediated radiofluorination and its further developments29−34 have improved the accessibility of such tracers. The application of the “low base” variant of this method allowed us to produce 6-[18F]FTrp in fair RCY of

IDO1-expressing tumor cells in vitro. Furthermore, Xin and Cai and Michelhaugh et al. observed accumulation of [18F]FETrp in different IDO1-positive tumor xenografts in mice.26,27 Unfortunately, the rather inefficient and cumbersome preparation procedure which includes enantioselective semipreparative HPLC and furnishes the probe in a RCY of only 6% within 90 min precludes the more widespread evaluation of this promising tracer. Application of conventional SNAr radiofluorination was only reported for the preparation of 4-[18F]FTrp.28 4-[18F]FTrp was synthesized in our group on a small scale (starting from 200−250 191

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15%.,34−36 Especially, our novel procedure of alcohol-enhanced Cu-mediated radiofluorination enabled the preparation of 18Flabeled indoles and model Trp derivatives in near quantitative radiochemical conversions (RCCs).32,35 Consequently, we used this protocol for the development of a simple and robust procedure for the preparation of (S)-4−7-[ 1 8 F]fluorotryptophans (4−7-[18F]FTrps, [18F]14−17) from the easily accessible aryl boronate precursors. The four PET tracer candidates were evaluated in different tumor cell lines (in vitro) and in healthy rats (in vivo). The most promising probe, 7[18F]FTrp, was additionally examined in a chicken chorioallantoic membrane xenograft model (CAM) of human cancers.

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RESULTS AND DISCUSSION Chemistry. First, the boronate precursors for radiolabeling were synthesized. The preparation of Nα,Nin-Boc2, O-tBuprotected (S)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)tryptophan (18) started with the alkylation of the (S)-Ni-BPBGly complex (19)37 with N,N,N-(4-bromoindolyl)methyltrimethylammonium salt 20.38 The corresponding 4-BrTrp substituted Ni complex (21) was obtained as a single (S,S)isomer in 63% yield (Figure 3). 20 was prepared from commercially available 4-bromoindole using a Mannich type reaction followed by quaternization with MeI. 21 was decomposed with 3 M HCl in 50% MeOH to give the crude 4BrTrp which was Nα-Boc and O-tBu-protected in two sequential steps. Boc-4-BrTrp-OtBu (22) was obtained in 39% yield over three steps. Noteworthy, only a single chromatographic purification was necessary to prepare 22 from 4-bromoindole. Alternatively, the Ni complex 21 was synthesized as a mixture of easily separable (S,S) and (S,R) diastereomers (2:1) in 41% yield by direct alkylation of 4-bromoindole with known (S)-Ni-BPBΔAla (23)39 using Et2AlCl as a catalyst. To the best of our knowledge this is the first example of Friedel−Crafts reaction using Ni complexes like 23 as alkylation agents. Finally, Nin-Boc protection of 21 followed by Miyaura borylation delivered the desired radiofluorination precursor 18. 5-BrTrp (24) was prepared from racemic Ac-5-BrTrp-OH [(RS)-25]40 via enzymatic deacetylation using acylase I from porcine liver in 20% yield (Figure 4).41 Subsequent N-Boc and O-tBu protection steps followed by Miyaura borylation furnished the respective pinacol boronate (Bpin) ester 26 in 47% yield over three steps. Alternatively, 24 was treated with AcOtBu in the presence of HClO4 affording Nin-tBu substituted amino ester 27. The latter was Nα-trityl protected affording after Miyaura borylation Trt-5-BpinTrp(tBu)-OtBu (28) in 44% yield over three steps. The known bis-lactim diether 2936 (Figure 4) was used for the preparation of 6-[18F]FTrp. Boc-7-BpinTrp-OtBu (30) was prepared in 56% yield from Boc-Trp-OtBu42 via Ir-catalyzed 2,7-diborylation followed by an in situ 2-deborylation according to the modified protocol of Loach et al. (Figure 5).43 Unlike many other species such as humans, mice, rabbits, and chickens, rats very efficiently transform (R)-Trp into the (S)isomer by enzymatic oxidation/reductive amination.44,45 Consequently, racemic [18F]FTrps could be presumably used instead of pure (S)-isomers for preclinical studies in rats. To prove this, precursors of racemic 6- and 7-[18F]FTrps were prepared. (RS)Boc-6-BpinTrp-OtBu [(RS)-31] was synthesized from (RS)Boc-6-BpinTrp-OEt [(RS)-32]32 in 67% over two steps as shown in Figure 5. (RS)-30 was prepared from Boc-(RS)-Trp-OtBu like 30.

Figure 5. Synthesis of radiolabeling precursors 30 and (RS)-32.

Radiochemistry. According to the original procedure for alcohol-enhanced Cu-mediated 18F-fluorination, 18F− was eluted from the anion exchange resin with a solution of Et4NHCO3 (2.7 mg, 14 μmol) in nBuOH (0.4 mL) into a vial containing a solution of the corresponding boronate precursor and Cu(py)4(OTf)2 (60 and 26.5 μmol, respectively) in DMA (0.8 mL; final concentration of alcohol 33%). After elution, the resulting solutions were directly heated at 110 °C for 5−20 min under air atmosphere furnishing radiolabeled aromatics and heteroaromatics in RCCs of up to 99%.32 The main drawback of this approach is the need for high precursor amounts. Consequently, we evaluated the dependency of RCCs on the precursor amount using N-Boc-5-Bpin-indole (33) as a model indole substrate (Figure 6; the amounts of Et4NHCO3 and Cu(py)4(OTf)2 were

Figure 6. Optimization of alcohol-enhanced Cu-mediated radiofluorination.

appropriately adjusted). With 30 and 20 μmol of precursor RCCs dropped down to 85 and 75%, respectively. In order to obviate the significant drop of RCCs and/or 18F− recovery, we switched to the elution of 18F− using Et4NHCO3 in MeOH according to Richarz et al.36,46 After elution, low boiling methanol was removed within 2−5 min at 70−80 °C, and the residue was taken up in a solution of precursor and Cu salt in 33% nBuOH in DMA and heated. This modification allowed us to achieve RCCs of 90% with only 10 μmol of precursor (Table 1). With the optimized radiolabeling procedure in hand we turned to the production of [18F]FTrps. N-Boc and O-tBu protected 5-, 6-, and 7-[18F]Trp were prepared in >90% RCCs using only 10− 15 μmol of the respective Bpin precursors (Figure 7). Similarly, an 18F incorporation yield of 87 ± 2% was observed for bis-lactim diether 29. Somewhat lower RCCs of 77 ± 12% and 70−95% were obtained using Boc-4-BpinTrp-OtBu (18) and Trt-5BpinTrp(tBu)-OtBu (28), respectively. These results presumably could be explained by intermolecular Chan-Lam reaction in the 192

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Figure 7. Preparation of 4−7-[18F]FTrps. aRCCs. bRCYs.

Table 2. Comparison of the Different Procedures for the Preparation of [18F]FTrps this work 40 ± 12 53 ± 5 30 ± 3a 42 ± 5b,c 41 ± 3 100−110 >99, 89a 95−240

18

4-[ F]FTrp (RCY %) 5-[18F]FTrp (RCY %) 6-[18F]FTrp (RCY %) 7-[18F]FTrp (RCY %) synthesis time (min) ee (%) molar activity (GBq/μmol) a

Atkins et al.21

Weiss et al.28

“low base”36

13

5 × 10−4

Tang et al.49

c, e

9−10c 9−10c

255

Giglio et al.48

115 >99 7 × 10−7

23 ± 8 15 ± 4a,d

8 12 4e

24 ± 5d 100−110 >99, 89a 95−240

9c,e ndi ndi ndi

1.5

193 73−82,f,g 98g,h 403−722

b

From bis-lactim precursor 29. From Boc-6-BpinTrp-OtBu. cRacemic. dThis work. eEstimated by the separation of a small portion of the reaction mixture. f5-(S)-[18F]FTrp; gAfter separation of the racemic mixture by preparative enantioselective HPLC. h5-(R)-[18F]FTrp. ind: no data.

final solutions determined by ICP/MS did not exceed 7 μg/ preparation and was well below any level of concern.47 During the preparation of this manuscript, two papers describing the low-yielding preparation of [18F]Trps via the original procedure for Cu-mediated radiofluorination were published (Table 2).48,49 Giglio et al. prepared 4-(RS)[18F]FTrp, 5-[18F]FTrp, 6-[18F]FTrp, and 7-(RS)-[18F]FTrp in RCYs of 8%, 12%, 4%, and 9%, respectively.48 Tang et al. prepared 5-[18F]FTrp and 5-(R)-[18F]FTrp in RCYs of 1.5% within 193 min with enantiomeric excess of 73−82% and 98%, respectively.49 Our previously reported “low-base” protocol34,36 delivered 5, 6-, and 7-[18F]FTrps also in lower RCYs of 23 ± 8%, 15 ± 4%, and 24 ± 5%, respectively (Table 2, column 4). In Vitro Study. High metabolic stability is one of the main prerequisites for the practical application of PET tracers as insufficient metabolic stability leads to increased accumulation of radioactivity in nontarget tissues lowering the image quality. All

first case and by the incomplete stability of the Trt protecting group under radiolabeling conditions in the second case. N-Boc, O-tBu protected radiolabeled intermediates were quantitatively hydrolyzed using 6 M HCl in 50% MeOH at 80 °C within 5 min. 12 M HCl at 110 °C was necessary to completely deprotect Trt-5-[18F]FTrp(tBu)-OtBu ([18F]39) within 15 min. [18F]40 was decomposed by heating with 50% H2SO4 at 130 °C for 15 min. Finally, 4-, 5-, 6-, and 7-[18F]FTrps were isolated by HPLC and obtained as ready-to-use solutions in 6−8% EtOH. Starting from N-Boc, O-tBu protected Bpin precursors [18F]FTrps were produced in 40−53% radiochemical yields (RCYs) within 100−105 min and with excellent radiochemical (>98%) and, if appropriate, enantiomeric purities (>99% ee). 6-[18F]Trp was prepared from 2936 in RCY of 30 ± 3% within 110 min and with 89% ee. Molar activities amounted to 34−79 GBq/μmol (1.5−3 GBq tracer). The Cu content in the 193

DOI: 10.1021/acs.jmedchem.7b01245 J. Med. Chem. 2018, 61, 189−206

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Next, the cellular uptake of 4−7-[18F]FTrps in different tumor cells lines was determined and compared with that of O-(2[18F]fluoroethyl)tyrosine ([18F]FET). [18F]FET is currently clinically used for the visualization of increased protein synthesis rate especially in brain tumors.52−54 [18F]FET and Trps are transported into tumor cells predominantly by type 1 L-amino acid transporters (LAT1) and trapped there presumably owing to the asymmetry of the intra- and extracellular recognition by LAT1. [18F]FET protein incorporation rate is very low.55 For cell uptake experiments, U-87 MG and U-138 MG glioblastoma, HD-MB03 medulloblastoma, PC3 and LNCaP C4-2 prostate as well as MDA-MB-231 (estrogen receptors positive) and MCF7 (estrogen receptors negative) breast tumor cell lines were selected. Noteworthy, the relatively low expression of IDO1 in U-87 MG, U-138 MG, and MDA-MB-231 cells can be stimulated by the incubation with interferon-γ (γITF) in a time-dependent manner. Expression of IDO in MCF7 cells remains very low even under γITF stimulation.24 In contrast, these cells express an increased level of tryptophan hydroxylase 1 (TPH1), an enzyme that catalyzes the rate-limiting Trp hydroxylation step of the serotonin biosynthesis.56 The cellular uptakes of 4−7-[18F]FTrps relative to those of 18 [ F]FET are shown in Table 3.57 U-87 MG and U-138 MG glioblastoma cells accumulated comparable amounts of radiolabeled fluorotryptophans and [18F]FET. In contrast, HD-MB03 cells had taken up considerably more [18F]FTrps than [18F]FET (up to 5−6× for 4- and 6-[18F]FTrps after 2 h of incubation). The uptake of 4−7-[18F]FTrps in PC3 and LNCaP C4-2 cells was about 2 times higher than that of [18F]FET. Whereas there was no significant difference in accumulation of [18F]FTrps and [18F]FET in untreated MDA-MB-231 cells, the uptake of 6- and 7-[18F]Trps (up to 2× and 7× after 2 h of incubation, respectively) was markedly increased after γITF stimulation. This effect was less pronounced in the cases of 4- and 5[18F]FTrps. [18F]FET uptake was only slightly altered after γITF stimulation. Uptake in MCF7 cells was about 2 times higher for 7-[18F]FTrp than for [18F]FET. In contrast, this cell line accumulated comparable amounts of 4-6-[18F]FTrps and [18F]FET.

four radiofluorinated tryptophans demonstrated a high stability in human blood serum at 37 °C. Only extremely low decomposition ( 9 and extracted with CH2Cl2 (×5). The organic fraction was washed with brine (×2), dried, and concentrated under reduced pressure. The residue was purified by column chromatography [MeOH/CH2Cl2 = 19:1 (0.1% Et3N)] followed by the low temperature recrystallization from CH2Cl2/hexane (−25 °C) furnishing 27 (0.43 g, 76%) as a colorless oil. Rf = 0.37 (MeOH/CH2Cl2 = 19:1); mp = 163 °C; 1 H NMR (600 MHz, DMSO-d6) δ 8.14 (br, 2H), 7.78 (d, J = 1.6 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.38 (s, 1H), 7.22 (dd, J = 8.9, 1.6 Hz, 1H), 4.18 (t, J = 6.8 Hz, 1H), 3.15 (ddd, J = 22.3, 14.9, 6.8 Hz, 1H), 1.64 (s, 4H), 1.29 (s, 4H); 13C NMR (151 MHz, DMSO-d6) δ 168.7, 133.4, 130.9, 127.3, 123.1, 121.1, 115.3, 111.4, 105.3, 82.7, 55.9, 52.9, 29.2, 27.3, 26.1. ESI HRMS: calcd for C19H27BrN2O2+, 397.13082; found, 397.13067. Trt-5-BrTrp(tBu)-OtBu. Et3N (0.335 mL, 0.243 g, 2.18 mmol) was added to a solution of 27 (0.43 g, 1.09 mmol) and trityl chloride (0.36 mg, 1.2 mmol) in CH2Cl2 (10 mL), and the reaction mixture was stirred 2 h. Afterward, H2O (20 mL) was added. Layers were separated, and the aqueous fraction was extracted with CH2Cl2 (×3). The combined organic fractions were washed with H2O (×2), brine (×2), dried, and concentrated under reduced pressure. The residue was purified by column chromatography (gradient 25 → 50% CH2Cl2 in petrol ether) to give the desired product (0.62 g, 89%) as a colorless solid. Rf = 0.4 (petrol ether/CH2Cl2 = 1:1); mp = 160 °C; 1H NMR (600 MHz, DMSO-d6) δ 7.69−7.60 (m, 2H), 7.41−7.14 (m, 17H), 3.32−3.28 (m, 1H), 2.94 (dd, J = 14.1, 7.1 Hz, 1H), 2.81 (d, J = 9.1 Hz, 1H), 2.74 (dd, J = 14.1, 6.1 Hz, 1H), 1.62 (s, 9H), 1.01 (s, 9H); 13C NMR (151 MHz, DMSO-d6) δ 173.1, 146.1, 133.3, 131.5, 128.4, 127.8, 126.8, 126.3, 122.8, 121.6, 115.1, 111.0, 108.4, 79.5, 70.8, 57.4, 55.6, 30.8, 29.3, 27.4. ESI HRMS: calcd for C38H41BrN2O2+, 639.24037; found, 639.24052. Elemental analysis calcd (%) for C38H40BrN2O2: C, 71.58; H, 6.86; N, 4.39. Found: C, 71.58; H, 6.86; N, 4.24. Trt-5-BpinTrp(tBu)-OtBu (28). 28 (0.38 g, 53%) as colorless foam was prepared according to GP 1 from Trt-5-BrTrp(tBu)-OtBu (0.67 g, 1.04 mmol). The crude product was purified by column chromatography (gradient 25 → 50% CH2Cl2 in petrol ether). Rf = 0.12 (petrol ether/CH2Cl2 = 1:1); mp = 93 °C; 1H NMR (600 MHz, DMSO-d6) δ 7.86 (s, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.50−7.00 (m, 5H), 2.78 (d, J = 9.0 Hz, 1H), 2.77 (ddd, J = 19.9, 14.0, 6.8 Hz, 1H), 1.64 (s, 2H), 1.30 (s, 3H), 1.03 (s, 2H); 13C NMR (151 MHz, DMSO-d6) δ 173.4, 146.3, 136.5, 129.2, 128.4, 127.8, 127.5, 126.8, 126.7, 126.4, 126.3, 125.4, 117.0, 112.6, 109.1, 83.1, 79.5, 70.6, 57.4, 55.4, 29.3, 27.5, 24.84, 24.82. ESI HRMS: calcd for C44H53BN2O4+, 685.41712; found, 685.41728. Boc-7-BpinTrp-OtBu (30). Pinacolborane (2.31 mL, 2.04 g, 15.92 mmol) was added to a solution of Boc-Trp-OtBu42 (1.14 g, 3.16 mmol), (1,5-cyclooctadiene)(methoxy)iridium(I) dimer (53 mg, 80 μmol) and 4,4′-di-tert-butyl-2,2′-dipyridyl (43 mg, 0.16 mmol) in anhydrous THF (32 mL) under glovebox conditions, the reaction flask was taken out of the glovebox, and the reaction mixture was stirred at 60 °C for 16 h. The

reaction mixture was concentrated under reduced pressure and dissolved in AcOH (4 mL), and Pd(OAc)2 (36 mg, 0.16 mmol) was added. The reaction mixture was stirred for 20 h at 30 °C and diluted with Et2O (50 mL). The resulting solution was washed with 10% NaHCO3 (3 × 20 mL), brine (3 × 20 mL), dried, and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc/hexane/CH2Cl2 = 5:22:2, silica with 0.1% CaO; two times) followed by low temperature recrystallization from pentane (two times) furnishing 30 (0.475 g, 31%) as a colorless solid. The chromatographic fractions containing Boc-3,7-diBpinTrp-OtBu (the spot directly over the product spot) and mother liquors from recrystallizations were collected together and concentrated. The residue was dissolved in AcOH (3.5 mL), and Pd(OAc)2 (36 mg, 0.16 mmol) was added. The reaction mixture was stirred for 24 h at 30 °C. Thereafter, workup and purification as described before afforded the second crop of 30 (0.4 g, 56% overall yield). Rf = 0.31 (EtOAc/hexane/ CH2Cl2 = 5:22:2); 1H NMR (300 MHz, CDCl3; mixture of two rotamers; only signals of the major rotamer are shown) δ 1.40 (s, 12 H), 1.41 (s, 9 H), 1.43 (s, 9 H), 3.18−3.38 (m, 2 H), 4.47−4.59 (m, 1 H), 5.04 (d, J = 8.76 Hz, 1 H), 7.07−7.12 (m, 1 H), 7.14 (t, J = 7.63 Hz, 1 H), 7.65 (d, J = 6.95 Hz, 1 H), 7.74 (d, J = 7.63 Hz, 1 H), 9.14 (br, 1 H); 13C NMR (75.48 MHz, CDCl3) δ 24.99, 27.76, 27.99, 54.73, 79.44, 81.70, 83.78, 110.00, 118.96, 122.60, 122.67, 126.90, 129.37, 141.19, 155.23, 171.37; signal of C-B was not observed. ESI HRMS: calcd for C26H40O6N2B+, 487.29743; found, 487.29756. (RS)-Boc-6-BpinTrp-OtBu [(RS)-31]. To a solution of (RS)-Boc-6BpinTrp-OEt [(RS)-32]32 (0.34 g, 0.74 mmol) in THF (10 mL) was added dropwise 1 M NaOH (0.74 mL) followed by H2O (until a homogeneous solution was obtained), and the reaction mixture was stirred for 1 h. Afterward, the second portion 1 M NaOH (0.7 mL) was added dropwise and the mixture was stirred for a further 10 h. Et2O and 1 M NaHSO4 (50 mL of each) were added, organic fraction was separated, washed with brine (2 × 10 mL), dried, and concentrated under reduced pressure. The residue was dissolved in anhydrous CH2Cl2 (10 mL), tert-butyl 2,2,2-trichloroacetimidate (0.27 mL, 0.324 g, 1.48 mmol) was added, and the reaction mixture was stirred at 45 °C for 24 h. Thereafter, the mixture cooled to ambient temperature, diluted with pentane (30 mL), and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by column chromatography (EtOAc/hexane = 1:3, silica with 0.1% CaO) followed by low temperature recrystallization from pentane affording (RS)-31 (0.24 g, 67% over two steps) as a colorless solid. Rf = 0.36 (EtOAc/hexane = 3:1); 1H NMR (200 MHz, CDCl3) δ 1.11−1.59 (m, 30 H), 3.15−3.35 (m, 2 H), 4.42−4.69 (m, 1 H), 5.09 (br, 1 H), 7.08 (s, 1 H), 7.47−7.67 (m, 2 H), 7.87 (s, 1 H), 8.42 (br, 1 H); 13C NMR (50.32 MHz, CDCl3) δ 24.86, 27.56, 27.90, 28.30, 54.71, 79.53, 81.78, 83.52, 110.63, 118.13, 118.32, 124.32, 125.19, 130.29, 135.77, 155.22, 171.30; signal of C-B was not observed. ESI HRMS: calcd for C26H39O6N2BK+: 525.25331; found, 525.25332; calcd for C26H40O6N2B+, 487.29743; found, 487.29743. Radiochemistry. General. All radiosyntheses were carried out using anhydrous DMA and nBuOH stored over molecular sieves (available from Acros or Aldrich). Commercially available substrate for radiolabeling 33 was used as received. Cu(OTf)2(py)4 was prepared according to the literature74 and stored under ambient conditions without any precautions. StrataX cartridges were obtained from Phenomenex (Aschaffenburg, Germany), and Sep-Pak Accell Plus QMA carbonate plus light cartridges, 46 mg sorbent per cartridge, were from Waters GmbH (Eschborn, Germany). [18F]Fluoride was produced by the 18O(p,n)18F reaction by bombardment of enriched [18O]water with 16.5 MeV protons using a MC16 cyclotron (Scanditronix, Sweden) at the Max Planck Institute for Metabolism Research (Cologne) or a BC1710 cyclotron (The Japan Steel Works Ltd., Japan) at the INM-5 (Forschungszentrum Jülich). All radiolabeling experiments were carried out under ambient or synthetic air. Each radiochemical experiment was carried out at least in triplicate. TLC Analyses. TLC analyses were carried out on TLC Al foils precoated with silica gel 50 mm × 100 mm with fluorescent indicator (silica gel 60 F254, Merck, Darmstadt) as follows. Reaction mixtures 201

DOI: 10.1021/acs.jmedchem.7b01245 J. Med. Chem. 2018, 61, 189−206

Journal of Medicinal Chemistry

Article

cartridges have a single frit on the male side but four frits on the female side]. 18F− was slowly eluted into the reaction vial using a solution of Et4NHCO3 (0.8 mg, 4 μmol) in MeOH (0.8 mL). MeOH was evaporated using a flow of air at 80 °C within 2 min. Afterward, the residual [18F]Et4NF was dried at the same temperature for 1−2 min. Using this procedure, recovery of 18F− amounted to 93 ± 4% (n > 30). In order to account for surface-adsorbed 18F−, reaction vials were completely emptied after the addition of water, and radioactivity in the solution and remaining radioactivity in the reaction vessel was separately determined and quantified. In all cases, ≥95% of radioactivity was observed in the solution. Synthesis of [18F]FET and 2-[18F]FDG. [18F]FET and 2-[18F]FDG were produced at the cyclotron facility of INM-5 (Forschungszentrum, Jülich) as described previously.75,76 Synthesis of Protected [18F]FTrps ([18F]35−38, [18F]39, [18F] 40): General Procedure 1 (GP1). A solution of the corresponding pinacol boronate precursor (7.5−15 μmol) and Cu(py)4(OTf)2 (7.5− 15 μmol; the same amount as of radiolabeling precursor) in DMA/ nBuOH 2:1 (750 μL) was added to [18F]Et4NF (0.1−20 GBq) (vice versa), and the resulting solution was heated under air at 110 °C for 15 min. Afterward, H2O (0.75 mL) was added and the reaction mixture was loaded onto C18 cartridge (500 mg, Waters, preconditioned with EtOH followed by H2O; 5 mL of each). The cartridge was washed with H2O (5 mL) and dried with air (20 mL). The crude protected radiofluorinated tryptophan was eluted with acetone (1.5−2 mL). Acetone was evaporated at 80 °C in a flow of air or He, affording radiolabeled intermediate which was deprotected according to the appropriate protocol (vide infra). Alternatively, the reaction mixture was concentrated at a flow of Ar at 110 °C and 400 mbar within 10 min and the residue was hydrolyzed using the appropriate protocol (vide infra). Deprotection of Boc-4-[18F]FTrp(Boc)-OtBu ([18F]35), Boc-5[18F]FTrp-OtBu ([18F]36), and Boc-6-(RS)-[18F]FTrp(Boc)-OtBu ([18F]37). Preparation of 4-, 5-, and (RS)-6-[18F]FTrps. The crude protected [18F]Trps was taken up in 12 M HCl/MeOH 1:1 (1 mL), and the resulting solutions were heated at 80 °C for 5−10 min and concentrated in a flow of He at the same temperature. The residue was dissolved in 7% EtOH, and radiolabeled amino acids were isolated by semipreparative HPLC (vide supra). Deprotection of Boc-7-[18F]FTrp(Boc)-OtBu ([18F]38). Preparation of 7-[18F]FTrp. [18F]38 was deprotected with 12 M HCl/ MeOH 1:1 (1 mL) at 60 °C for 5 min, and 7-[18F]FTrp was isolated as described above. Deprotection of [18F]38. Preparation of 5-[18F]FTrp (Alternative Route). [18F]39 was deprotected with 12 M HCl (0.75 mL) at 110 °C for 15 min, the reaction mixture was cooled to ambient temperature and diluted with 7% EtOH (2 mL), and the resulting solution was injected into the HPLC system. 5-[18F]FTrp was isolated by semipreparative HPLC as described above. Deprotection of [18F]39. Preparation of 6-[18F]FTrp. [18F]41 was taken up in NMP (0.25 mL), 50% H2SO4 (0.75 mL v/v) was added, and the reaction mixture was heated at 130 °C for 15 min. The reaction mixture was cooled to ambient temperature, diluted with 9% EtOH (4 mL), and the resulting solution was loaded into the HPLC system. 6[18F]FTrp was isolated by semipreparative HPLC as described above. Molar Activity Calculation. The molar activities (GBq/μmol) were calculated by dividing the radioactivity of the 18F-labeled product by the amount of the unlabeled tracer determined from the peak area in a UV−HPLC chromatograms (λ = 254 nm). The amounts of unlabeled compounds were determined from the UV absorbance/concentration calibration curve. The solutions of [18F]FTrps obtained after HPLC purification were concentrated under reduced pressure, and the residues were redissolved in the appropriate HPLC eluents (500 μL). The resulting solutions were completely injected into the HPLC system. The peak area was determined, and the amount of carrier was calculated according to the calibration curve. Determination of Serum Stability of [18F]FTrps. A solution of the corresponding purified [18F]FTrp (100−150 MBq;